Liquid tissue preparation from histopathologically processed biological samples, tissues and cells

ABSTRACT

The current invention provides a method for directly converting histopathologically processed biological samples, tissues, and cells into a multi-use biomolecule lysate. This method allows for simultaneous extraction, isolation, solublization, and storage of all biomolecules contained within the histopathologically processed biological sample, thereby forming a representative library of said sample. This multi-use biomolecule lysate is dilutable, soluble, capable of being fractionated, and used in any number of subsequent experiments.

This application claims priority to Provisional Application No.60/452,956, filed on Mar. 10, 2003, the contents of which are herebyincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention provides methods of processing histopathologicallyprocessed biological samples, tissue, and cells into a biomoleculelysate that is suitable for multiple uses. The methods allow extraction,isolation, solubilization, and storage of biomolecules from the lysates,including proteins, glycoproteins, nucleic acids, lipids, glycolipids,and cell organelle-derived molecules. This multi-use biomolecule lysateis soluble, dilutable, capable of being fractionated, and usable in anynumber of subsequent biochemical assays.

BACKGROUND OF THE INVENTION

For over a hundred years, public and academic medical universities andinstitutions, pathology clinics, private biomedical institutions, tissuearchives, hospitals, and museums have been preserving biologicalspecimens with formalin and other chemical fixatives such asformaldehyde and ethyl alcohol. The most common chemical fixative isformalin. Formalin is used as a fixative because of its superior abilityto preserve both tissue structure and cellular morphology. This hasresulted in the wide use of formalin for the successful preservation ofhistologic sections for traditional microscopic analysis. Formalinfixation is so effective in preserving tissue structure and cellularmorphology that the formalin archive is a veritable treasure trovecontaining millions of samples. Within this archive are biologicalsamples of healthy tissue, tissue samples from virtually every knowndisease, and a multitude of preserved life forms.

The most common form of sample fixation occurs through formalin-inducedcross-linking of the proteins within the biological specimen. Theseprotein cross links, while providing excellent cellular morphologypreservation, also renders the fixed sample relatively insoluble.Because of these protein cross-links, the types of assays that can beperformed on a formalin-fixed sample are limited in number, unable toprovide quantitative results and lack sensitivity. In fact, formalinfixed biological samples are virtually unusable in many modern assaytechniques, which are both highly quantitative and sensitive.

It is apparent, therefore, that new methods for solubilizingformalin-fixed or other chemically-fixed biological samples are greatlyto be desired.

SUMMARY OF THE INVENTION

An object of the present invention provides for a method to solubilizeformalin-fixed biological samples. More specifically, the presentinvention provides for a method of obtaining a multi-use biomoleculelysate from a histopathologically processed biological sample.

In accordance with one aspect of the invention there is provided amethod of preparing a multi-use biomolecule lysate, comprising the stepsof: (a) heating a composition comprising a histopathologically processedbiological sample and a reaction buffer at a temperature and a timesufficient to negatively affect protein cross-linking in the biologicalsample, and (b) treating the resulting composition with an effectiveamount of a proteolytic enzyme for a time sufficient to disrupt thetissue and cellular structure of the biological sample.

The histopathologically processed biological sample may comprise asubstantially homogeneous population of tissues or cells. The sample maybe, for example, formalin-fixed tissue/cells, formalin-fixed/paraffinembedded (FFPE) tissue/cells, FFPE tissue blocks and cells from thoseblocks, and/or tissue culture cells that have been formalin fixed and/orparaffin embedded.

If the sample is embedded in paraffin or some similar material, theparaffin may be removed by, for example, adding an organic solvent,heating; heating and adding a buffer comprising Tris, and/or heating andadding an organic solvent. Advantageously this step is carried out priorto the main heating step. If the sample is heated as part of the processto remove paraffin, the heating need only be brief, for example a fewminutes. This brief heating advantageously may be repeated two or moretimes to ensure maximum removal of paraffin.

At any stage, the sample may be mechanically disrupted by, for examplemanual homogenization; vortexing; and/or physical mixing. The lysateproduced by these methods may be subjected to a wide variety ofbiochemical assays. The lysate also may be fractionated, for exampleinto nucleic acid and protein fractions, before assay. Each biomoleculefraction typically contains distinct and separate biomolecules that aresuitable for use in biochemical assays.

The heating step may be carried out, for example, at a temperaturebetween about 80° C. and about 100° C. and for a period of from about 10minutes to about 4 hours. The proteolytic enzyme treatment lasts, forexample, for a period of time from about 30 minutes to about 24 hours.The proteolytic enzyme treatment may be carried out, for example, at atemperature between about 37° C. to about 65° C. In each step, thereaction buffer may comprise a detergent, and/or a detergent may beadded after the protease treatement. The detergent may be, for example,Nonidet P40, SDS, Tween-20, Triton X, and/or sodium deoxycholate,although the skilled artisan will recognize that other detergents may beused. The proteolytic enzyme may be for example, proteinase K,chymotrypsin, papain, pepsin, trypsin, pronase, and/or endoproteinaseLys-C, although the skilled artisan will recognize that other enzymesmay be used. The reaction buffer may comprise Tris and may have a pH inthe range of about 6.0 to about 9.0.

It is a further object of the invention to provide a kit for preparing amulti-use biological lysate, where the kit contains (a)histopathologically processed biological sample, (b) a proteolyticenzyme, and (c) a detergent.

It is yet another object of the invention to provide methods ofdetecting one or more analytes in a multi-use biomolecule lysatesuspected of containing the one or more analytes, comprising the stepsof: (a)contacting a multi-use biomolecule lysate as described above withan array, where the array comprises one or more capture agents of knownbinding specificity immobilized on a support surface in a positionallydistinguishable manner; and (b) detecting the binding or absence ofbinding of one or more analytes in the lysate to the immobilized capturereagents. One or more of the analytes may be, for example, a protein.The capture reagents may be, for example, antibodies and antibodyfragments, single domain antibodies, engineered scaffolds, peptides,nucleic acid aptamers, a receptor moiety, affinity reagents, smallmolecules such as, for example, drugs, and protein ligands, althoughother capture reagents also could be used. The support surface may be,but is not limited to, for example, a material selected from the groupconsisting of glass, derivitized glass, silicon, derivitized silicon,porous silicon, plastic, nitrocellulose membranes, nylon membranes, andPVDF membranes. One or more of the analytes may be a nucleic acid or anucleic acid, such as RNA or DNA. The multi-use biomolecule lysate maybe subjected to a fractionation step prior to contacting the lysate withthe array.

It is a still further object of the invention to provide methods ofanalyzing two or more multi-use biomolecule lysates obtained from two ormore histopathologically processed biological samples, comprising thesteps of (a) immobilizing two or more multi-use biomolecule lysatesobtained from a histopathologically processed sample on a supportsurface, where each lysate is immobilized at a discrete location on thesurface; (b) contacting the support surface with a reagent of knownbinding affinity; and (c) detecting the presence or absence of bindingof the reagent of known binding affinity at the discrete locations onthe support surface. In a particular embodiment, the detecting step (b)may be carried out using a detection reagent that specifically binds toone or more of the analytes suspected to be present in the sample. Thelysate may be fractionated prior to immobilization on the surface. Forexample, the RNA, DNA, and/or protein fractions of the lysate may beimmobilized on the surface.

The lysate may be spotted onto the support surface by, for example,manual spotting, ink-jetting, robotic contact printing, roboticnon-contact printing and/or piezoelectric spotting. The reagent of knownbinding affinity may be, for example, an antibody or antibody fragments,a single domain antibody, an engineered scaffolds, a peptide, a nucleicacid aptamer, a receptor moiety, an affinity reagent and/or a proteinligand. The support surface may be, but is not limited to, for example,a material selected from the group consisting of glass, derivitizedglass, silicon, derivitized silicon, porous silicon, plastic,nitrocellulose membranes, nylon membranes, and PVDF membranes.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be better understood with reference to theappended drawing sheets wherein:

FIG. 1 is a flow chart illustrating the method of the multi-usebiomolecule lysate preparation of the present invention.

FIG. 2 shows a typical protein expression analysis and demonstrates thata multi-use biomolecule lysate preparation is dilutable and can be usedfor quantitative protein expression profiling of cells obtained fromformalin fixed tissue samples.

FIG. 3 shows a standard multi-use biomolecule lysate preparation thatwas fractionated. Both fractions were recovered and used for gelelectrophoresis analysis for nucleic acid determination.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for treating histopathologicallyprocessed biological samples in a manner that allows the samples to beused in a wide variety of biochemical assays. For example, the methodsof the invention permit for the first time the recovery of proteins andnucleic acids from histopathologically processed biological samples in aform that is useful for further assays.

Specifically, the present inventors have surprisingly found thathistopathologically processed biological samples can be heated in areaction buffer, followed by protease treatment, to provide lysates thatare rich in molecular information regarding the original biologicalsample. Vast numbers of histopathologically processed biological samplesfrom a huge array of normal and diseased tissues are available inlaboratories and hospitals around the world, and the methods of thepresent invention expand to a significant degree the information thatcan be obtained from those samples, as described in more detail below.

It is to be understood that the present invention is not limited to theparticular methodologies, protocols, constructs, formulae and reagentsdescribed and as such may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention.

As used herein and in the appended claims, the singular forms “a,”“and,” and “the” include plural reference unless the context clearlydictates otherwise. Thus, for example, reference to “a cell” is areference to one or more cells and includes equivalents thereof known tothose skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devices,and materials similar or equivalent to those described herein can beused in the practice or testing of the invention, the preferred methods,devices and materials are now described.

All publications and patents mentioned herein are incorporated herein byreference for the purpose of describing and disclosing, for example, thecompositions and methodologies that are described in the publicationsthat might be used in connection with the presently described invention.The publications discussed above and throughout the text are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing herein is to be construed as an admission that theinventor is not entitled to antedate such disclosure by virtue of priorinvention.

Biological Samples

The present invention provides for a method of obtaining a multi-usebiomolecule lysate from a histopathologically processed biologicalsample.

Histopathologically processed biological samples can include wholeorganisms, samples obtained from diagnostic surgical pathology, tissuesamples, body fluids, cellular or viral material, or any otherbiological sample that has been histopathologically processed. Uses ofthe multi-functional lysate include diagnostic or predictive diseasemodeling, but this lysate may also be used in conjunction with anyuseful laboratory technique as dictated by the particular circumstances.

One embodiment of the present invention provides for obtaining amulti-use biomolecule lysate from a histopathologically processedbiological sample. Examples of histopathological processing ofbiological samples include, but are not limited, to: formalin fixationof whole organisms; formalin fixation of tissues or cells; formalinfixation/paraffin embedding of tissues or cells; and formalin fixationand/or paraffin embedding of tissue culture cells.

Histopathological processing typically occurs through the use of aformalin fixative. Formalin is used widely because it is relativelyinexpensive, easy to handle, and once the formalin-fixed sample isembedded in paraffin the sample is stored easily. Additionally, formalinis often the fixative of choice because it preserves both tissuestructure and cellular morphology. Although the exact mechanism may notbe understood fully, fixation occurs by formalin-induced cross-linkingof the proteins within the biological specimen. Due to these proteincross-links, formalin fixation has found wide success in the traditionalmicroscopic analysis of histologic sections. Once a biological sample ishistopathologically processed however, it is no longer soluble. As aresult, only a few experimental techniques are available forhistopathologically processed biological samples. The current assaysthat can be performed on a formalin-fixed sample are both few and atbest, semi-quantitative. Examples of assays that can be performed onformalin fixed tissue are immunohistochemistry (IHC), in situhybridization (ISH), and fluorescence in situ hybridization (FISH). ISHand FISH provide cellular localization of mRNA or DNA. These assays allsuffer from the same shortcomings in terms of lack of quantification,low sensitivity, and difficulty in performing high-throughput assays.Formalin fixation therefore renders the formalin fixed archive of littlevalue for many of the powerful analysis methods that have been developedin recent years.

The sheer volume of formalin-fixed specimens cannot be overstated. Fornearly the last one hundred years, biological specimens have beencommonly fixed in formalin or formalin fixed/paraffin wax-embedded(FFPE) blocks. Universities and museums have vast archives of plants andanimals that are formalin-fixed. Hospitals, in the course of diagnosticsurgical pathology, have established large formalin-fixed collectionsthat contain tissues from nearly every known disease in addition tonormal, healthy tissue. Due to the need to retain these clinical tissuesamples in case further testing is required, these archives around theworld now contain millions of FFPE samples.

One embodiment of the invention described herein provides for thecreation of a soluble multi-use biomolecule lysate fromhistopathologically processed biological samples. This method includesmaking a multi-use biomolecule lysate directly from ahistopathologically preserved biological sample, e.g., a tissue or cell,allowing one to obtain, extract, isolate, solubilize, fractionate, andstore substantially all of the biomolecules of various types containedwithin the sample. This soluble multi-use biomolecule lysate forms arepresentative library of all of the biomolecules as they existed withinthe histopathologically processed biological sample. Such biomoleculesinclude but are not limited to proteins, glycoproteins, nucleic acids(e.g., DNA, RNA), lipids, glycolipids, and cell organelle-derivedmolecules.

In addition, the multi-use biomolecule lysate is malleable. For example,the multi-use biomolecule lysate may be fractionated into a nucleic acidfraction and a fraction that contains the remaining biomolecules bymethods well known in the art. Furthermore, this multi-use biomoleculelysate is capable of being serially diluted. Another embodiment of thepresent invention provides for a method wherein a multi-use biomoleculelysate from a histopathologically processed biological sample may beused in a number of experimental techniques.

Uses of the Lysates

The method described herein is particularly useful because it can beused to obtain a multi-use biomolecule lysate from a histopathologicallyprocessed biological sample capable of being used with numerousexperimental and diagnostic techniques, thereby providing new uses forthe histopathologically processed archive. Examples of techniques thatthe multi-use biomolecule lysate can be used with include but are notlimited to are chromatography, protein arrays, Western blotting,immunoprecipitation, affinity columns, alternative splicing assays,mutation analysis, nucleic acid amplification, labeled probes formicroarray analysis, RFLP analysis, Southern blotting, andhigh-throughput assays such as but not limited to one- andtwo-dimensional polyacrylamide gel electrophoresis (2D-PAGE), serialanalysis of gene expression (SAGE), HPLC, FPLC, MALDI-TOF massspectroscopy, SELDI mass spectroscopy, liquid chromatography, massspectrometry, ELISA assays, Quantitative RT-PCR, Single NucleotidePolymorphism detection, genotyping and sequencing. The skilled artisanwill recognize that the lysates produced by the methods of the inventionalso may be used in a wide variety of additional assays.

The recent completion of the Human Genome Project, in addition tospurring dramatic advances in the field of genomics and proteomics, hasdemonstrated the vast potential of high throughput assays. Proteomicshas gone beyond its initial attempts to identify and quantify proteinsand is now attempting to determine the functions of all proteins in anorganism, organ, or organelle, and how these proteins vary with time andphysiological state.

“Functional genomics” attempts to determine the physiological role ofeach gene. An important step in discovering the function of each gene isto carefully measure the expression patterns of MRNA transcripts andproteins in tissue specimens. By measuring specific expression patternsof genes and gene products such as mRNA and proteins, one can determinewhat genes are expressed and at what levels in a normal, healthy celltype. Perhaps more importantly however, is that by measuring theexpression patterns in diseased cell types, new insight will be gleanedinto the pathological progression of that disease. In addition, newmarkers may be discovered, thereby yielding new diagnostic andtherapeutic strategies.

The ability to utilize the formalin fixed archive would be a tremendousaid in these undertakings. Often times, patient outcome is known foreach pathological specimen. Correlations between markers and patientprognosis therefore could be readily created. In addition, because themulti-use biomolecule lysate is a representative library of thehistopathologically processed biological sample, both nucleic andnon-nucleic fractions are present. Therefore, direct relationshipsbetween nucleic acid expression and the presence of non-nucleic acidmolecules can be determined. This is an advantage over currenttechniques that isolate only the nucleic acid fraction or thenon-nucleic acid fraction and only indirect correlations may be drawn.Alternatively, high-throughput assays are a boon to comparative andevolutionary biologists and zoologists due to the ability of theseassays to generate and quantify differences between species. The scopeof these undertakings is only possible through the use ofhigh-throughput assays.

Array Assays

A specific example of a high-throughput assay is the protein array.Protein arrays are highly parallel (multiplexed) and can be conducted inminiature (microarray). Protein arrays are quick, usually automated,highly sensitive, and capable of generating an enormous amount of datain a single experiment. The protein array is essentially a miniaturizedversion of the familiar ELISA or dot blotting immunoassay. Similar toELISA and dot blots, protein array results are usually obtained usingoptical detection methods, for example fluorescent detection. The datagenerated by a single protein array experiment often is so voluminousthat specialized computer software may be required to read and analyzethe data that is generated.

High-throughput assays such as protein array analysis are capable ofscreening vast amounts of biological samples at once. In order tosignificantly link a single candidate marker to any disease, a largenumber of cases must be screened to generate definite correlations.However, obtaining enough biological samples with known disease outcomesin a frozen or stable and storable state that is not chemically fixed isa limiting factor. A possible solution to this limitation would be theuse of the archive of formalin-fixed tissues and cells. The methods ofthe current invention are particularly useful because the multi-usebiomolecule lysate allows the formalin-fixed tissue archive to be usedin high-throughput protein array analysis.

In one type of protein array analysis, specific capture reagents ofknown binding affinity, such as antibodies, are immobilized or spottedonto a support surface in a known positional manner, thus forming theprotein array. Plasma, other tissue extracts, or in this case themulti-use biomolecule lysate is then added to the protein array. Becausethe immobilized binding proteins on the support surface have a specificaffinity for an individual protein or marker, protein arrays are able todetect target molecules or marker proteins in the specimen. Byimmobilizing the specific capture reagents in known locations on thesupport surface, protein identification and the presence of markerproteins can be determined by x, y positional information. In addition,since differences in protein levels within complex samples can be easilymeasured, accurate quantitative differential analysis can also beperformed. Detection is achieved through a number of methods known tothose well versed in the art. Examples include but are not limited to:secondary antibodies in sandwich assays, direct labeling of analytes,dual color labeling, mass spectrometry, surface plasmon resonance, andatomic force microscopy.

An alternative type of protein array analysis places tissue/cell lysatesin an arrayed format, for example on a solid support. Multiple lysatesfrom different samples may be arrayed on a single surface in apositionally identifiable manner. Reagents of known binding specificity,such as antibodies, that bind to target biomolecules or markers are thenadded. The main difference between the two major types of array analysesdescribed herein is that in the first type of protein array, theexpression of many different proteins across a single source of protein(a single cancer tissue for example) can be determined. In contrast, bythe other type of protein array analysis, one can assay for theexpression of one protein at a time across many different sources ofprotein (many different cases of cancer tissues for example). The lysatemay be fractionated prior to immobilization on the array, and proteincontaining fractions of the lysates may be used to prepare the array.The skilled artisan will recognize also that other fractions of thelysates can be used to prepare arrays. For example, DNA and/or RNAcontaining fractions can be immobilized on suitable surfaces to preparenucleic acid arrays.

Specific reagents of known binding affinity in protein arraysadvantageously are antibodies or antibody fragments, but may also besingle domain antibodies, engineered scaffolds, peptides, nucleic acidaptamers, small molecules such as drugs, for example, protein ligands,or other specific binding proteins known in the relevant art. Antibodiesmay be either polyclonal or monoclonal, or a portion or fragment of anantibody capable of binding antigenic sites, and are available from theusual commercial sources such as Sigma-Aldrich Co. (St. Louis, Mo.).

Protein array support surfaces include but are not limited to glass(such as slides), silicon, porous silicon, nylon, PVDF or nitrocellulosemembranes and the like, or beads and are available from a variety ofcommercial sources. Alternatively, specialized chips have been developedfor protein assays and are commercially available from, for example,Biotrove (Woburn, Mass.), Zyomyx (Hayward, Calif.) and Pontilliste(Mountain View, Calif.).

Specific capture reagents or tissue/cell lysates can be spotted orimmobilized onto the support surface by a number of techniques familiarto those knowledgeable in the arts. Examples include, but are notlimited to, robotic contact printing, robotic non-contact printing, inkjetting, and piezoelectric spotting. If the capture reagent is a polymerthat may be synthesized on a solid support, such as a nucleic acid, thecapture reagent may be prepared directly on the support by, for example,photolithography. A number of automated commercial spotters areavailable from, for example, Packard Bioscience (Meriden, Conn.) andmanual spotting equipment also is commercially available from, e.g. V &P Scientific (San Diego, Calif.).

As used herein, the term “analyte” refers to a biomolecule containedwithin the biological sample that is detectable by binding to a reagentof specific binding affinity.

As used herein, the term “buffer” refers to buffer which has a specificpH in the range of 1.0 to 9.0. Both specific pH and buffer types areselected based upon the proteolytic enzyme used. Both buffer type andspecific pH requirements are known to those well versed in the arts.

As used herein, the term “organic solvent” refers to solvents forremoving paraffin including but not limited to xylene, toluene, orchloroform.

As used herein, the term “incubate” refers to bringing a reagent ofknown binding affinity in contact with the biological sample in order tofacilitate binding between the reagent of known specific bindingaffinity and analytes contained in the biological sample. Incubationtime and reagent concentration need only be sufficient to obtain adesired result, although the skilled artisan will recognize that bothincubation time and reagent concentration may be optimized using methodsthat are known in the art once suitable reagents have been identified.

As used herein, the term “sufficient homogeneity” refers to a populationof tissues or cells that possess similar characteristics or traits basedon selection criteria. An example of selection criteria includes but isnot limited to histopathological selection criteria that are well knownin the relevant arts. Examples of methods of actually “obtaining” abiological sample include but are not limited to using a manual corepunch, tissue punch, laser microdissection and other techniques that arewell known in the arts. The actual size of the obtained biologicalsample is not important as long as there is a sufficient amount toperform the chosen assay.

Methods of Preparing Lysates

The methods of the present invention involve heating thehistopathologically processed biological sample for a time and at atemperature sufficient to negatively affect the formalin-induced proteincross-links. The skilled artisan will recognize that time andtemperature of heating are not critical and may be varied, though atypical period for heating is from about 30 minutes to about 24 hours.The mechanisms by which temperature may negatively affect theformalin-induced cross-links include but are not limited to reversing ofthe protein cross-links. Although the exact mechanism is not known, andwithout being bound by any theory, the present inventors believe thatthe negative affect of temperature on the protein cross-links appears toinvolve some form of releasing, reversing, or partial modification ofthe cross-links.

The method of the present invention further involve adding at least oneproteolytic enzyme to the histopathologically processed biologicalsample. Proteolytic enzymes are believed to augment the negative effectof heating on formalin-induced protein cross-links. The time,temperature, and quantity of the proteolytic enzyme are not critical aslong as they are all sufficient to negatively affect theformalin-induced protein cross-links. Examples of proteolytic enzymesthat are suitable for use in the present invention include but are notlimited to trypsin, proteinase K, chymotrypsin, papain, pepsin, pronase,and endoproteinase Lys-C. Trypsin may be purchased from Sigma-Aldrich(St. Louis, Mo.). Advantageously, the protease treatment is carried outfollowing the heating step described above. The protease treatment isadvantageously carried out at a temperature that is optimal for maximumactivity of the protease, but this can be varied.

In one embodiment of the current invention, the multi-use biomoleculelysate may be fractionated into distinct and separate biomolecules thatmay be collected separately. Examples of biomolecule fractions that canbe collected include but are not limited to protein, glycoproteins,nucleic acids (e.g., DNA, RNA), glycolipids, and lipids. Fractionationtechniques are well known in the arts and include but are not limitedto, spin column fractionation, immunoprecipitation, gradientcentrifugation, HPLC and drip column fractionation. Other fractionationmethods are well known in the art.

After fractionation, the desired fraction of the multi-use biomoleculelysate may be used in subsequent assays. Examples of assays thatbiomolecule fractions of the multi-use biomolecule lysate can be usedwith include but are not limited to are column chromatography, proteinarrays, Western blotting, immunoprecipitation, affinity columns,alternative splicing assays, mutation analysis, nucleic acidamplification (for example PCR, LCR, and T7 based RNA amplification),labeled probes for microarray analysis, RFLP analysis, Southernblotting, and high-through put assays such as but not limited to one-and two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), serialanalysis of gene expression (SAGE), HPLC, FPLC, MALDI-TOF massspectrometry, SELDI mass spectrometry, liquid chromatography, massspectrometry, ELISA assays, Quantitative RT-PCR, Single NucleotidePolymorphism detection, genotyping and sequencing.

The present invention includes articles of manufacture, such as “kits.”Such kits will typically be specially adapted to contain in closecompartmentalization each container holding a component useful incarrying out the preparation of the multi-use lysate according to themethods and compositions taught herein. In a particular embodiment, thepresent invention provides compositions that contain ahistopathologically processed biological sample, a reaction buffer, anda detergent. The kit may further include a protease, and a reagent forremoving paraffin from the sample, such as a buffer and/or an organicsolvent

FIG. 1 is a flow chart illustrating an embodiment of the presentinvention, comprising a method of preparing the multi-use biomoleculelysate and subsequently utilizing said lysate in a number of differentassays. These “steps” need not be performed in any particular order, andserve as a non-limiting description as follows:

-   -   (a) applying specific selection criteria, based on histology, to        a biological sample to achieve an enrichment of specific        homogeneous biological tissue/cell populations. The enrichment        can be carried out, for example, by tissue microdissection        methods, before biomolecule procurement in the form of liquid        tissue preparation;    -   (b) adding a specific pH-adjusted (ranging from pH 6.0 to pH        9.0) Tris-based buffer to said procured biological sample for        stabilization of peptides, peptide fragments, proteins, enzymes,        DNA, DNA fragments, RNA, RNA fragments, and other biomolecules        and biomolecule fragments;    -   (c) imparting some level of physical disruption to the        biological sample by a method that includes but is not limited        to manual homogenization, vortexing, and/or physical mixing;    -   (d) heating the biological sample at an elevated temperature in        the range of from about 80° C. to about 100° C. for a period of        time from about 10 minutes to about 4 hours. Temperature range        and time period may be determined by those of skill in the art,        based for example, on sample size;    -   (e) adding one or more proteolytic enzyme(s) including for        example proteinase K, chymotrypsin, papain, pepsin, trypsin,        pronase, and endoproteinase Lys-C to the biological sample for a        period of time from about 30 minutes to about 24 hours at an        elevated temperature from about 37° C. to about 80° C.,        advantageously at a temperature from about 37° C. to about        65° C. The temperature range and time may be determined by        skilled artisans considering, for example, the size of the        biological sample and/or the chosen proteolytic enzyme;    -   (f) adding one or more detergents including for example        Nonidet-P-40, SDS, Tween-20, Triton-X, and sodium deoxycholate        to the biological sample. The detergent may be added prior to        the protease treatment step (before or after heating), in which        case the nature of the detergent and its concentration is        selected so as to not substantially inhibit the activity of the        protease, or may be added after the protease step;    -   (g) molecularly fractionating the resulting biological sample by        some method as for example spin column fractionation,        immunoprecipitation, gradient centrifugation, HPLC, and drip        column fractionation in order to separate specific molecular        fractions, resulting in the procurement of different        biomolecules in different and collectable fractions;    -   (h) procuring and purifying specific subcellular and molecular        fractions for the procurement of different biomolecules for        subsequent biochemical assays.

In the present invention, each lysate of biomolecules forms arepresentative library of specific biomolecules that directly reflectsthe status of those biomolecules as they previously resided in thehistopathologically processed biological sample. An example of such arepresentative biomolecular lysate library from histopathologicallyprocessed biological sample would be the preparation of a lysate ofproteins directly from formalin-fixed paraffin embedded tissue/cells.

The resulting preparation of biomolecules can be placed in an arrayedformat for the simultaneous biochemical analysis of multiple liquidtissue preparations (multi-use biomolecule lysate) obtained frommultiple and different histopathologically processed biological samples.An example of such a high throughput array assay format would be thedevelopment of a liquid tissue protein array such that tissue proteinlysates derived from histopathologically processed samples and procuredas stated above are arrayed in an ordered and defined pattern as smallspots of protein on a solid support substrate, where each spot is arepresentative library of the expressed proteins, and characteristics ofthose expressed proteins, that resided in the histopathologicallyprocessed biological sample, and that when assayed by a number ofvarious biochemical protein analysis formats, such as immuno-basedprotein identification binding assays, do directly reflect theexpression pattern and characteristic of the proteins as they relate tothe pathology and histology of the histopathologically processedbiological sample from which the proteins were procured.

When the biomolecule of interest is a protein, the protein extract is ina soluble liquid form and is representative of the total protein contentof the cells procured from the starting histopathologically processedbiological sample. The protein extract can be placed in any number ofprotein identification, analysis and expression assays including but notlimited to liquid tissue protein microarrays that contain representativelibraries of proteins from pathologically and histologically definedpopulations of histopathologically processed biological sample and asthese analyses relate to the histology, disease state, and pathology ofthe histopathologically processed biological sample.

When the biomolecule is DNA, the DNA extract is in a soluble liquid formand is representative of the total DNA content of the cells procuredfrom the starting histopathologically processed biological sample. TheDNA extract can be placed in any number of DNA and/or RNA geneidentification analyses and monitoring assays designed to determinevariations in DNA including but not limited to the analysis of genestructure, genetic variability, single nucleotide polymorphisms andmutation analyses as these analyses relate to the histology, diseasestate, and pathology of the histopathologically processed biologicalsample.

When the biomolecule is RNA, the RNA extract is in a soluble liquid formand is representative of the total RNA content of the cells procuredfrom the starting histopathologically processed biological sample. TheRNA extract can be placed in any number of RNA and/or geneidentification analysis and gene expression analysis and quantitativeRT-PCR analysis as these analyses relate to the pathology, diseasestate, and histology of the starting histopathologically processedbiological sample.

When the biomolecule is a biomolecule other than protein, DNA and RNA,the biomolecule is assayed as it relates to the pathology, disease stateand histology of the starting histopathologically processed biologicalsample.

The present invention, thus generally described, will be understood morereadily by reference to the following examples, which are provided byway of illustration and are not intended to be limiting of the presentinvention.

EXAMPLE 1 Preparation of a Multi-Use Lysate from a Formalin-Fixed Sample

1. Place one 2 mm diameter by 25 μm thick section from a tissue punchinto a silanized or low protein binding 1.5 ml microcentrifuge tube.

2. Add 500 μl of 20 mM Tris-HCl pH 7.8.

3. Heat at 95° C. for 1 minute.

4. Mix gently on a vortex mixer.

5. Carefully, without disturbing the tissue section, remove the bufferusing a pipettor.

6. Add 750 μl of 20 mM Tris-HCl pH 7.8.

7. Heat at 95° C. for 1 minute.

8. Carefully, without disturbing the tissue section, remove the bufferusing a pipettor.

9. Microcentrifuge at 10,000 rpm for 1 minute.

10. Remove any residual buffer from the microcentrifuge tube with apipettor.

11. Add 10 μl of reaction buffer (10 mM Tris-HCl pH 7.8, 1.5 mM EDTA,0.1% Triton X-100, 10% glycerol) to the tube. Make sure that the tissueis at the bottom of the tube and covered with reaction buffer.

12. Heat at 95° C. for 1.5 hours. Every 20 minutes, check the tube andshake the buffer that has formed a condensation in the cap down to thebottom of the tube so that it covers the tissue section before placingthe tube back into the heating block.

13. Microcentrifuge at 10,000 rpm for 1 minute.

14. Place tubes on ice to cool.

15. Add 0.5 μl of 1% Trypsin and gently mix.

16. Incubate for 1 hour at 37° C. Every 20 minutes check the tube andshake the buffer that has formed a condensate in the cap down to thebottom of the tube. Vortex rigorously for 10 to 15 seconds. Shake thebuffer down to the bottom of the tube so that it covers the tissuesection before placing the tube back into the waterbath.

17. Microcentrifuge at 10,000 rpm for 1 minute.

18. Heat at 95° C. for 5 minutes.

19. Microcentrifuge at 10,000 rpm for 1 minute.

The resulting multi-use biomolecule lysate may be either used insubsequent assays or stored at −20° C. until ready for use.

While there has been described what is presently believed to be thepreferred embodiments of the present invention, other and furthermodifications and changes may be made without departing from the spiritof the invention. We intend to include all further and othermodifications and changes that come within the scope of the invention asset forth in the claims.

1. A method of preparing a multi-use biomolecule lysate, comprising thesteps of: (a) heating a composition comprising a histopathologicallyprocessed biological sample and a reaction buffer at a temperature and atime sufficient to negatively affect protein cross-linking in saidbiological sample, and (b) treating the resulting composition with aneffective amount of a proteolytic enzyme for a time sufficient todisrupt the tissue and cellular structure of said biological sample. 2.The method according to claim 1, wherein said histopathologicallyprocessed biological sample comprises a substantially homogeneouspopulation of tissues or cells.
 3. The method according to claim 1,further comprising, prior to step (a), the step of removing any paraffinpresent in said histopathologically processed biological sample by oneor more methods selected from the group consisting of: adding an organicsolvent; heating; heating and adding a buffer comprising Tris; andheating and adding an organic solvent.
 4. The method according to claim1, further comprising the step of mechanically disrupting saidbiological sample by at least one technique selected from the groupconsisting of: manual homogenization; vortexing; and physical mixing. 5.The method according to claim 1, wherein said biological sample isheated to a temperature between about 80° C. and about 100° C.
 6. Themethod according to claim 1, wherein said biological sample is heatedfor a period of time from about 10 minutes to about 4 hours.
 7. Themethod according to claim 1, wherein said proteolytic enzyme treatmentlasts for a period of time from about 30 minutes to about 24 hours. 8.The method according to claim 1, wherein said proteolytic enzymetreatment is carried out at a temperature between about 37° C. to about65° C.
 9. The method according to claim 1 wherein said reaction buffercomprises a detergent.
 10. The method according to claim 1 wherein step(b) is carried out in the presence of a detergent.
 11. The methodaccording to claim 9, wherein said detergent is selected from the groupconsisting of Nonidet P40, SDS, Tween-20, Triton X, and sodiumdeoxycholate.
 12. The method according to claim 10, wherein saiddetergent is selected from the group consisting of Nonidet P40, SDS,Tween-20, Triton X, and sodium deoxycholate.
 13. The method according toclaim 1, wherein said proteolytic enzyme is selected from the groupconsisting of proteinase K, chymotrypsin, papain, pepsin, trypsin,pronase, and endoproteinase Lys-C.
 14. The method according to claim 1,wherein said reaction buffer comprises Tris and has a pH in the range ofabout 6.0 to about 9.0.
 15. The method according to claim 1 furthercomprising the step of fractionating said multi-use biomolecule lysateinto distinct and separate biomolecule fractions.
 16. The methodaccording to claim 15 wherein each biomolecule fraction containsdistinct and separate biomolecules suitable for use in biochemicalassays.
 17. The method according to claim 1, wherein saidhistopathologically processed biological sample is selected from a groupconsisting of formalin-fixed tissue/cells, formalin-fixed/paraffinembedded (FFPE) tissue/cells, FFPE tissue blocks and cells from thoseblocks, and tissue culture cells that have been formalin fixed and orparaffin embedded.
 18. A kit for preparing a multi-use biological lysatecomprising at least: (a) histopathologically processed biological sample(b) a proteolytic enzyme, and (c) a detergent
 19. A method of detectingone or more analytes in a multi-use biomolecule lysate suspected ofcontaining said one or more analytes, comprising the steps of: (a)contacting a multi-use biomolecule lysate according to claim 1 with anarray, wherein said array comprises one or more capture agents of knownbinding specificity immobilized on a support surface in a positionallydistinguishable manner; and (b) detecting the binding or absence ofbinding of one or more analytes in said lysate to said immobilizedcapture reagents.
 20. The method according to claim 19, wherein at leastone of said analytes is a protein.
 21. The method according to claim 20,wherein said capture reagent is selected from the group consisting ofantibodies and antibody fragments, single domain antibodies, engineeredscaffolds, peptides, nucleic acid aptamers, a receptor moiety, affinityreagents, small molecules, and protein ligands.
 22. The method accordingto claim 20, wherein the support surface comprises a material selectedfrom the group consisting of glass, derivitized glass, silicon,derivitized silicon, porous silicon, plastic, nitrocellulose membranes,nylon membranes, and PVDF membranes.
 23. A method of analyzing aplurality of multi-use biomolecule lysates obtained from a plurality ofhistopathologically processed biological samples, comprising the stepsof (a) immobilizing a plurality of multi-use biomolecule lysatesobtained from a histopathologically processed sample on a supportsurface, wherein each lysate is immobilized at a discrete location onsaid surface; (b) contacting said support surface with a reagent ofknown binding affinity; (c) detecting the presence or absence of bindingof said reagent of known binding affinity at said discrete locations onsaid support surface.
 24. The method according to claim 23, wherein themulti-use biomolecule lysate is spotted onto the support surface by amethod selected from the group consisting of manual spotting,ink-jetting, robotic contact printing, robotic non-contact printing andpiezoelectric spotting.
 25. The method according to claim 23, whereinsaid reagent of known binding affinity is selected from the groupconsisting of antibodies and antibody fragments, single domainantibodies, engineered scaffolds, peptides, nucleic acid aptamers, areceptor moiety, affinity reagents, small molecules, and proteinligands.
 26. The method according to claim 23, wherein said supportsurface comprises a material selected from the group consisting ofglass, derivitized glass, silicon, derivitized silicon, porous silicon,plastic, nitrocellulose membranes, nylon membranes, and PVDF membranes.27. The method according to claim 19 wherein at least one of saidanalytes is a nucleic acid.
 28. The method according to claim 27,wherein said multi-use biomolecule lysate is subjected to afractionation step prior to contacting said lysate with said array. 29.The method according to claim 27, wherein said nucleic acid comprisesRNA.
 30. The method according to claim 27, wherein said nucleic acidcomprises DNA.
 31. The method according claim 23, wherein theRNA-containing fraction of each lysate is immobilized on said supportsurface.
 32. The method according to claim 19, wherein the detectingstep (b) is carried out using a detection reagent that specificallybinds to one or more of the analytes suspected to be present in saidsample.
 33. The method according to claim 32, wherein said detectionreagents are proteins.
 34. The method according to claim 23, wherein oneor more of said multi-use biomolecule lysates is subjected to afractionation step prior to immobilizing one or more of the resultingfractions on said surface.
 35. The method according to claim 34, whereinthe nucleic acid fraction of one or more lysates is immobilized on saidsurface.
 36. The method according to claim 35, wherein said nucleic acidfraction is an RNA-containing fraction.
 37. The method according toclaim 35, wherein said nucleic acid fraction is a DNA-containingfraction.
 38. The method according to claim 34, wherein said fraction isa protein-containing fraction.
 39. The method according to claim 1,further comprising the step of subjecting the product of step (b) to atleast one biochemical assay.